Impaired wound healing, susceptibility to skin breakdown and skin fragility lead to skin infections and considerable morbidity and mortality in the US. Our recent study shows that while no significant difference in epidermal stem cell frequency could be detected with aging, the proliferative capacity of individual epidermal stem cells was reduced. Also, transit amplifying cell frequency was greater in the aged, cell cycle duration was increased, and premature progenitor differentiation was observed. Our HYPOTHESIS is that Bmi-1 has a key role in the altered differentiation, proliferation, and symmetric divisions of aged EpiSCs and that intrinsic (Bmi-1) and/or extrinsic (niche) alterations can partially or completely restore the alterations in Bmi-1 expression, differentiation, proliferation, and symmetric division. The long-term goal of this project is to understand the mechanisms that underlie the alterations in epidermal stem cells and their niche with aging. This knowledge will be used to improve wound healing in the aged and for tissue engineering applications. SPECIFIC AIMS: In this proposal we will study the roles of both intrinsic (Bmi-1/p16) and extrinsic/environmental influences on epidermal stem cell aging. In Aim 1 we will optimize and complete our assay of the differences between aged and young EpiSCs and then investigate whether the epidermal stem cells of Bmi-1 knockout mice show a premature aging skin phenotype, (premature differentiation, decreased proliferation, extended cell cycle, increased symmetric division and increased p16 expression). We will then overexpress Bmi-1 in aged epidermal stem cells to determine whether the aging phenotype can be rejuvenated with Bmi-1 overexpression. In Aim 2 we will determine the effects of extrinsic factors, both local and systemic, on the aging of epidermal stem cells, and whether the premature differentiation, decreased proliferative capacity, increased cell cycle, increased symmetric division and decreased Bmi-1 can be reversed with a young local and/or systemic environment. These studies will be performed in a transplantation assay that allows us to look at the effects of local and systemic factors on individual progenitor cells. SIGNIFICANCE: Modifying age-related functional changes in EpiSCs through alteration of Bmi-1/p16 status could be used for retention of proliferative potential and suppression of terminal differentiation. Also better understanding environmental influences on EpiSCs would determine potential therapies to improve aged epidermal function. Great benefits for the elderly would include preventing skin breakdown/fragility (e.g. bedsores, bruising, tearing) and improved treatment of wounds (leg ulcers and slow healing surgical wounds). PUBLIC HEALTH RELEVANCE: Impaired wound healing, susceptibility to skin breakdown and skin fragility lead to skin infections and considerable morbidity and mortality in the US. In this proposal we will study the roles of both intrinsic (Bmi- 1/p16) and environmental influences on epidermal stem cell aging. Modifying age-related functional changes in EpiSCs through alteration of Bmi-1/p16 status could be used for retention of proliferative potential and suppression of terminal differentiation. Also better understanding environmental influences on EpiSCs would determine potential therapies to improve aged epidermal function. Benefits for the elderly would include preventing skin breakdown/fragility (e.g. bedsores, bruising, tearing) and improved treatment of wounds (leg ulcers and surgical wounds).